Voltage-dependent G-protein regulation of CaV2.2 (N-type) channels

How G proteins inhibit N-type, voltage-gated, calcium-selective channels (CaV2.2) during presynaptic inhibition is a decades-old question. G proteins Gβγ bind to intracellular CaV2.2 regions, but the inhibition is voltage dependent. Using the hybrid electrophysiological and optical approach voltage-clamp fluorometry, we show that Gβγ acts by selectively inhibiting a subset of the four different CaV2.2 voltage-sensor domains (VSDs I to IV). During regular “willing” gating, VSD-I and -IV activations resemble pore opening, VSD III activation is hyperpolarized, and VSD II appears unresponsive to depolarization. In the presence of Gβγ, CaV2.2 gating is “reluctant”: pore opening and VSD I activation are strongly and proportionally inhibited, VSD IV is modestly inhibited, while VSD III is not. We propose that Gβγ inhibition of VSDs I and IV underlies reluctant CaV2.2 gating and subsequent presynaptic inhibition.


INTRODUCTION
N-type voltage-gated calcium channels Ca V 2.2 are found at the presynaptic terminal of neurons in the central and peripheral nervous systems, where their activation initiates calcium-dependent neurotransmitter release (Fig. 1A) (1).Ca V 2.2 channels are renowned for their abundance in nociceptors and their role in the development and treatment of chronic pain (1).Neurotransmitters and neuromodulators such as noradrenalin, serotonin, γaminobutyric acid (2), and opioids (3) can inhibit Ca V 2.2.This was initially demonstrated in 1978 in dorsal root ganglion (DRG) neurons (2) and was eventually ascribed to G-protein-induced inhibition of Ca V 2.2 (1,4).The Gβγ complex directly inhibits Ca V 2.2 opening, reducing calcium influx and subsequent neurotransmitter release (Fig. 1B) (1).
The Ca V 2.2 α 1B pore-forming subunit consists of four interlinked repeats (I to IV) that form a central calcium-conducting pore domain (transmembrane segments S5 and S6 from each repeat) and four voltage-sensor domains (VSDs; segments S1 to S4 from each repeat) that surround and control the opening of the pore (Fig. 1, C and D) (5)(6)(7).The four VSDs differ in amino acid composition and likely respond differentially to depolarization (8,9).Binding sites for Gβγ have been identified in the N terminus, repeat I-II loop, and C terminus of Ca V 2.2 (Fig. 1C) (10)(11)(12).Curiously, G-protein inhibition is voltage dependent.As described by Bean (4), G-protein inhibition changes calcium-channel gating from willing to reluctant, shifting voltage-dependent activation toward depolarized potentials.G proteins can also inhibit Ca V 2.2 gating currents (13)(14)(15).Collectively, this points to an important role of the VSDs.Here, we present voltage-clamp fluorometry (VCF) data illuminating the activation of individual voltage sensors and their inhibition by G proteins-fundamental events controlling calcium-mediated transmitter release.
VSD I activation and pore opening appeared to be coupled, as observed by the closely overlapping fluorescence deflection-voltage (ΔF-V, blue) and tail current-voltage (I tail -V, black) curves (Fig. 2,  B and C).Specifically, VSD I activated with a half-activation potential V 0.5 = 4.7 ± 2.6 mV and had an apparent voltage sensitivity z = 3.0 ± 0.33 e 0 (n = 5).This was comparable to the pore opening of this construct (V 0.5 = 0.091 ± 2.6 mV, z = 2.8 ± 0.19 e 0 , n = 5).
VSD II did not respond to voltage changes.We tested three, uniformly spaced, labeling positions in the S3 II -S4 II linker: I567C, G570C, and F573C (Fig. 1E).Despite high functional expression, no ΔF could be observed, even at extreme voltages (−160 to 160 mV; Fig. 2A and fig.S1).To prevent any occurrence of stable quenching, we removed a nearby tryptophan (W564F), as in a previous VCF study on BK channels (22), but this made no difference (fig.S1).We also tested labeling with fluorophores with a longer stalk, 6-TAMRA C6 maleimide (23) or Alexa Fluor 488 C 5 maleimide, but this did not generate any ΔF (fig.S1).Initial structures of Ca V 2.2 hinted that S4 II might be stabilized in the down state by a PIP 2 molecule (6, 7), and PIP 2 is known to modulate Ca V 2.2 (24).Thus, we depleted PIP 2 using the voltage-sensitive phosphatase DrVSP, as previously described (25).PIP 2 depletion was confirmed as DrVSP-expressing cells had a significant reduction in I tail after a depolarizing pulse, compared to cells that did not express DrVSP; still, we did not observe ΔF (fig.S2).Subsequent structures of the Ca V 2.3 channel also showed S4 II in a down state, in the absence of PIP 2 (26,27), suggesting that the "locked down" S4 II is a feature of the Ca V 2 family independently of PIP 2 .
VSD III activated at notably negative potentials and had a halfactivation potential of −62.8 ± 5.6 mV, and low apparent voltage sensitivity at 1.1 ± 0.1 e 0 (n = 7) (Fig. 2, B and C).Hence, ~50% of Ca V 2.2-VSD III would be active at resting membrane potentials.At −20 mV, 4% of channels were open, while 90% of VSD III were activated.Last, VSD IV had a half-activation potential of −5.9 ± 1.5 mV, close to that of pore opening, and a low apparent voltage sensitivity of 1.6 ± 0.2 e 0 (n = 12) (Fig. 2, B and C).

VSD I responds more strongly than VSDs III and IV to pre-pulse facilitation
With the additional dimension of individual voltage-sensor resolution now available from VCF, we explored the mechanistic details of G-protein inhibition of Ca V 2.2.To recapitulate Ca V 2.2 inhibition by G proteins in our experimental paradigm, we coexpressed the Ca V 2.2 complex (α 1B /α 2 δ-1/β 2a ) together with the human Gβ1 and Gγ2 subunits.A hallmark of Gβγ inhibition is "pre-pulse facilitation" (28,29).This describes the current increase observed following a strongly depolarizing pre-pulse during Gβγ inhibition and is thought to be mediated by the transient unbinding of Gβγ and relief of Gβγ inhibition (29).When we optically tracked Ca V 2.2 VSD activation in the presence of Gβγ, we found that the VSDs facilitated to a different extent (Fig. 3): VSD I had the most prominent facilitation, VSD III did not respond to pre-pulse facilitation, and VSD IV had an intermediate response.This revealed that G-protein-coupled receptor (GPCR) signaling modulates the Ca V 2.2 VSDs, in a VSDselective manner.

Gβγ makes VSDs I and IV "reluctant" to activate
To further determine Gβγ inhibition of the VSDs, we studied their voltage-dependent activation in the absence or presence of Gβγ.To prevent the relief of Gβγ inhibition (dependent on time and voltage), we used shorter (20 ms) activation pulses.As expected, Gβγ shifted the V 0.5 of Ca V 2.2 pore opening toward positive potentials, by 14 ± 0.8 mV (P = 3 × 10 −7 ; Fig. 4, A and B).We found that VSD I activation is strongly inhibited by Gβγ.Gβγ shifted the VSD I V 0.5 by 21 ± 1.0 mV, P = 8 × 10 −8 , similar to channel opening in the same cells (Fig. 4, C and D).To evaluate whether the effects on VSD I activation were mediated via the canonical Gβγ binding sites, we introduced point mutation R54A, previously shown to abolish G-protein inhibition (30).Gβγ inhibition of both the pore opening and the VSD I activation was eliminated (fig.S3).The R54A mutation appeared to produce a depolarizing shift in the voltage dependence of both VSD I activation and channel opening.A plausible interpretation of this effect is that R54 electrostatically opposes VSD I deactivation, effectively facilitating VSD I activation and, consequently, channel opening.In contrast to VSD I, VSD III was not modulated by Gβγ, ΔV 0.5 = −0.1 ± 5.3 mV, P = 0.99 (Fig. 4, C and D).VSD IV displayed modest inhibition by Gβγ compared to VSD I, ΔV 0.5 = 13 ± 1 mV, P = 1 × 10 −6 (Fig. 4, C and D).
VSD I activation and pore opening are strongly and proportionately inhibited by Gβγ during DRG action potentials Ca V 2.2 is the predominant synaptic calcium channel in nociceptors (31)(32)(33)(34) and is important in pain sensitivity and the development of chronic pain (35)(36)(37).To investigate VSD operation under a physiological stimulus, we implemented an action potential clamp with a waveform characteristic of small, unmyelinated nociceptive DRG neurons (34,38,39).At the resting membrane potential (V rest , −60 mV) channels were closed (Fig. 5, A and B).
In the case of VSD I-labeled channels, the peak macroscopic conductance was observed during the plateau phase of the action potential, reaching 17 ± 1% of the maximum, as normalized by a standard "I tail -V" protocol on the same cell.In the presence of Gβγ, the peak conductance was significantly reduced by approximately half, to 8 ± 2% (P = 0.004; Fig. 5C).At V rest , VSD I was not activated, while already 60 ± 4% of VSD III and 14 ± 4% of VSD IV were active (Fig. 5, A and B)-assuming that limiting VSD activation was 100% and the fluorescence signal tracked VSD activation in an all-or-nothing manner.These fractions did not significantly change in the presence of Gβγ (VSD I: P = 0.33; VSD III: P = 0.88; VSD IV: P = 0.21).
Peak VSD activation was defined as the maximum amplitude of the ΔF, normalized by a standard "ΔF-V" protocol.Similar to pore opening, VSD I reached 24 ± 1% activation and was significantly reduced to 13 ± 3% in the presence of Gβγ (P = 0.007; Fig. 5).VSD III reached maximal activation independently of Gβγ (−Gβγ; 98 ± 10%; +Gβγ: 94 ± 5%; P = 0.4).Last, 53 ± 4% of VSD IV activated at peak, which was not significantly different in the presence of Gβγ (40 ± 5%; P = 0.09).To better illustrate the timing of VSD activations and pore opening, the activity traces were used to annotate the Ca V 2.2 structure in movie S1.

DISCUSSION
Our most recent knowledge on the VSDs of Ca V 2.2 came from the atomic structures resolved by cryogenic electron microscopy, effectively at 0 mV (6,7).However, the dynamic responses of these structures to electrical signals and their regulation by G proteins were unknown.Our optical investigation of the Ca V 2.2 VSDs under physiologically relevant conditions revealed that these domains exhibit distinct voltage-sensing properties and regulation by G proteins.In summary, we found that (i) VSDs I, III, and IV activate with distinct voltage dependencies, likely contributing in different ways to channel opening; (ii) in our tested conditions, VSD II does not respond to changes in the membrane potential; and (iii) Gβγ sets VSDs I and IV in a reluctant mode of activation.
The Ca V 2.2 voltage-sensing apparatus VSD I activation and pore opening exhibit very similar voltage dependence (Figs. 2, 4, and 5), suggesting that this domain is strongly coupled to pore opening.In favor of this interpretation, action potential clamp protocols revealed that VSD I activation and pore opening are coupled in both time and voltage (Fig. 5 and movie S1).Both achieved maximal activation or open probability of roughly 20% during an action potential.Introducing a negative charge in Ca V 2.2 S3 I , which might inhibit VSD I activation, shifted V 0.5 of pore opening by more than +10 mV (40).
VSD II may not be pertinent to channel opening, as discussed below.VSDs III and IV both activate within physiologically relevant potentials.VSD III, in particular, appears to be tuned to sensing the resting membrane potential.Voltage-clamp recordings and stimulation with an action potential waveform showed that 60% of VSD III is already active at resting membrane potential when channels are closed (Figs. 2, 4, and 5).This anticipates that Ca V 2.2 channels have at least two closed states: one with VSD III in the resting conformation and another with VSD III in the active conformation.This suggests that VSD III activation does not directly drive pore opening.VSD IV has a voltage dependence approaching that of pore opening, although the latter saturates at more negative potentials than VSD IV activation (Fig. 2).One interpretation is that VSDs I, III, and IV are all required for opening of the pore, where VSD III activation is the first VSD to transition to an active state and VSDs I and/or IV are limiting factors for pore opening.
We did not observe any fluorescence deflections from VSD II despite testing multiple positions and conditions, even at extreme levels of depolarization, hyperpolarization, and expression (Fig. 2 and figs.S1 and S2).Our findings, together with recent structural evidence, support the role of the VSD II alternative to voltage sensing.In 2021, two independent studies both revealed Ca V 2.2 channels with S4 II in a down state in the absence of an electric field (0 mV), while S4 I , S4 III , and S4 IV were in an up state (6,7).Later structures of the closely related Ca V 2.3 channel also demonstrated resting VSD II states (26,27).Moreover, Ca V 2.3 channels can undergo voltagedependent opening upon neutralization of S4 II gating charges, with only a modest shift in V 0.5 (27).VCF has revealed that VSD II does undergo voltage-dependent activation in Ca V 1 channels (8, 9), and accordingly, S4 II in resolved structures of Ca V 1 and Ca V 3 channels is in an active conformation (41)(42)(43).We propose that a voltageinsensitive VSD II is a signature of Ca V 2 family channels, where it may serve as a static structural element of the channel.Perhaps under different regulatory regimes or in other splice variants, VSD II can undergo voltage-dependent activation and contribute to Ca V 2.2 opening and regulation.

GPCR tuning of Ca V 2.2 voltage dependence
We found that VSD I is strongly inhibited by Gβγ, in proportion to pore-opening inhibition, while VSD IV is modestly affected and VSD III is not (Figs. 4 and 5).Accordingly, VSD I activation is also facilitated most strongly by pre-pulse facilitation, while VSD IV shows more modest facilitation and III is unaffected (Fig. 3).This hierarchy of VSD modulation by Gβγ suggests that Gβγ directly interacts with VSD I and potentially VSD IV, to prevent activation and subsequent opening.In single-channel recordings, Gβγ inhibition manifests as the emergence of low-open-probability (P O ), reluctant openings and delayed high-P O openings (44).Consolidating this information and our results, we propose that Gβγ acts by inhibiting VSD I-driven channel opening (delaying high-P O events), allowing the emergence of inefficient openings by VSD IV (low-P O events).The previously known Gβγ binding sites may position Gβγ in close proximity to VSD I (Fig. 1C) (10)(11)(12).Gβγ-mediated stabilization of the VSD I resting conformation may be due to electrostatic interactions.Introducing a negative charge in S3 I (G177E) to inhibit VSD I activation resulted in similar effects as G-protein inhibition: Ca V 2.2 opening becomes intrinsically reluctant and sensitive to pre-pulse facilitation (40).Neutralizing the R2 gating charge of S4 I in the closely related Ca V 2.1 channel also reduced G-protein inhibition (45).An interaction with VSDs I and IV in a down state could account for the preferential closed-state G-protein inhibition of Ca V 2.2 (46,47).
In conclusion, we show that Ca V 2.2 VSDs respond differently to voltage and that this asymmetry extends to their modulation by other proteins.Gβγ, the result of GPCR signaling in the presynaptic terminal, specifically acts by preventing the activations of VSDs I and IV.
Cells were held at −80 mV and stepped to a range of voltages for 50 ms when characterizing VSDs, or 20 ms when investigating G-protein modulation.In experiments investigating G-protein modulation, a pre-pulse facilitation protocol was used to confirm Gβγ modulation (28,29).Cells were held at −100 mV and the level of facilitation was determined at a 0-mV test pulse before and after a conditioning 100-ms pre-pulse at 100 mV.In cells co-injected with Gβγ, only cells displaying ≥50% facilitation were included in the data analysis.
Action potential waveform was derived from a model based on recordings in rat DRG neurons at room temperature (38) in MATLAB R2022a (MathWorks) using the ode15s differential equation solver.In action potential clamp experiments, cells were held at −60 mV, as this was the cell resting potential.
The Ca V 2.2 tail current I tail was obtained from the peak tail current, fitted to a Boltzmann equation where V is the membrane potential, I tail,max is maximal I tail , z is the valence, V 0.5 is half-activation potential, and R and F are the gas and Faraday constants, respectively.I tail (V ) = I tail,max ∕ 1 + exp zF V 0.5 − V ∕ (RT) (1)

Fig. 1 .
Fig.1.Ca V 2.2 function, G-protein inhibition, and structure.(A) the arrival of an action potential to the presynaptic terminus triggers the activation of ca v 2.2 channels, ca 2+ influx, and neurotransmitter release (1).(B) Neurotransmitters and neuromodulators can activate their cognate, presynaptic G-protein-coupled receptors (GPcRs) triggering G-protein hydrolysis and release.the Gβγ complex directly inhibits the voltage-dependent opening of ca v 2.2, reducing ca 2+ influx and neurotransmitter release (1).(C) Membrane topology of the ca v 2.2 pore-forming subunit (α 1B ). it comprises four homologous repeats, each spanning the membrane six times.the first four transmembrane segments of each repeat (S1 to S4) assemble into a distinct voltage-sensor domain (vSd), while S5 and S6 from all repeats form the central pore.Gβγ is thought to bind to cytosolic loop regions (magenta): the N terminus, the repeat i-ii loop, and the c terminus.(D) Structure of a human ca v 2.2 channel(6).(E) Amino acid sequence diversity among the four ca v 2.2 repeats.Positively charged residues (blue) drive vSd activation upon membrane depolarization.Pink boxes show cysteine-substituted positions for fluorescence labeling and voltage-clamp fluorometry.(F) Ribbon structures of vSds i to iv(6).Pink residues show cysteine-substituted positions used for site-directed fluorescent labeling.cobalt-blue residues are positively charged arginines (R) and lysines (K).Gray residues are phenylalanines (F) indicating the charge transfer center.Red residues are negatively charged countercharges (cc) in S2.Note the differences in gating charges R1-K6 and that vSd ii was resolved in a down state (resting, gating charges below F).